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Received 15 Aug 2013 | Accepted 30 Jan 2014 | Published 24 Feb 2014

Hsueh-Cheng Chiang1,*, Wonchul Shin1,*, Wei-Dong Zhao1, Edaeni Hamid1, Jiansong Sheng1, Maryna Baydyuk1, Peter J. Wen1, Albert Jin2, Fanny Momboisse1,w & Ling-Gang Wu1

Vesicle fusion with the plasma membrane generates an O-shaped membrane prole. Its pore is thought to dilate until attening (full-collapse), followed by classical endocytosis to retrieve vesicles. Alternatively, the pore may close (kiss-and-run), but the triggering mechanisms and its endocytic roles remain poorly understood. Here, using confocal and stimulated emission depletion microscopy imaging of dense-core vesicles, we nd that fusion-generated

O-proles may enlarge or shrink while maintaining vesicular membrane proteins. Closure of fusion-generated O-proles, which produces various sizes of vesicles, is the dominant mechanism mediating rapid and slow endocytosis within B130 s. Strong calcium inux triggers dynamin-mediated closure. Weak calcium inux does not promote closure, but facilitates the merging of O-proles with the plasma membrane via shrinking rather than full-collapse. These results establish a model, termed O-exoendocytosis, in which the fusion-generated O-prole may shrink to merge with the plasma membrane, change in size or change in size then close in response to calcium, which is the main mechanism to retrieve dense-core vesicles.

DOI: 10.1038/ncomms4356

Post-fusion structural changes and their roles in exocytosis and endocytosis of dense-core vesicles

1 National Institute of Neurological Disorders and Stroke, 35 Convent Drive, Building 35, Room 2B-1012, Bethesda, Maryland 20892, USA. 2 National Institute of Biomedical Imaging and Bioengineering (NIBIB), Bethesda, Maryland 20892, USA. * These authors contributed equally to this work. w Present address: Centro Interdisciplinario de Neurociencia de Valparaso, Universidad de Valparaso, Gran Bretana 1111, Playa Ancha, Valparaso 2360102, Chile. Correspondence and requests for materials should be addressed to L.-G.W. (email: mailto:[email protected]

Web End [email protected] ).

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Exocytosis, a process that involves the fusion of a vesicle with the plasma membrane to release vesicular contents, is crucial for many biological events, including brain activities

and endocrine functions1. To maintain exocytosis, fused vesicles are recycled via endocytosis. There are at least two exo endocytosis modes. One involves vesicle full-collapse (FC) into the plasma membrane and spread of vesicular membrane proteins2,3, followed by classical endocytosis involving membrane invagination and ssion to recycle vesicles46. The other mode, called kiss-and-run (KR)7,8, involves fusion pore opening and closure46. Although these two modes have been widely accepted, the presumed structural changes have not been observed in live cells at sub-diffraction-limited resolution. Here we used confocal and stimulated emission depletion microscopy (STED)9 at B230 nm and 90 nm resolution, respectively, to resolve these hypothesized structural changes in live chromafn cells containing B300 nm dense-core vesicles.

Although decades of studies in chromafn cells contribute to forming the current FC/KR theory4,6,10,11, the physiological role and the trigger mechanism of KR remain unresolved4,10. KR, which was rst detected as capacitance ickers in B0.2 s1214, was proposed to underlie an entire cells endocytosis15. However, endocytosis recorded from whole-cell capacitance measurements (whole-cell endocytosis) lasts for B2030 s15. This long time course is difcult to be accounted for by KR as brief as B0.2 s. A slower form of KR, termed cavicapture, has been revealed through imaging vesicular membrane proteins15. However, cavicapture is not considered the dominant mechanism mediating whole-cell endocytosis15. In brief, KR has not been established as the dominant endocytic mode in chromafn cells. Neither has it been established in other cell types46. Two factors might contribute to this situation. First, to assess the contribution of KR to whole-cell endocytosis, it is best to reconstruct the overall endocytosis from individual KR for comparison with the simultaneously recorded whole-cell endocytosis. This comparison is labour intensive because KR is not trivial to record13,15. Second, the stimulation condition and the mechanism that predominantly triggers KR remain elusive46,16. KR is proposed to be triggered by low calcium1719. Paradoxically, higher calcium inux speeds up whole-cell endocytosis in chromafn cells20,21 and other cell types2224.

In the present work, we develop an imaging technique with high spatial and temporal resolution to visualize structural changes of fusion-generated O-shaped membrane prole while simultaneously monitoring whole-cell endocytosis with capacitance measurements in chromafn cells. We nd that O-prole does not dilate as predicted by the FC model or simply close its pore as predicted by the KR model, but dynamically changes in seven modes. These results establish a model that redenes FC and KR fusion as part of a larger spectrum of structural changes, with varied triggers and physiological roles. We suggest to redene KR as rapid or slow closure of O-shaped prole, during which this prole may change in size before closure, resulting in various sizes of vesicles. Pore closure is mediated by dynamin and triggered by large calcium inux. It is the predominant mechanism mediating whole-cell rapid and slow endocytosis lasting for B130 s. It may also mediate bulk endocytosis, a form of endocytosis that generates large vesicles5,6. Low calcium inux facilitates shrinking of the fusion-generated O-shaped prole, leading to the merge of the fused vesicle with the plasma membrane. These results call for substantial modication of the classical FC and KR model.

ResultsImaging X-prole at the instant of fusion. To label O-shaped membrane proles and assess its structural changes, primary

cultured bovine chromafn cells were bathed with membrane-impermeable uorescent dyes (Fig. 1a)25. With Alexa Fluor 647 (A647, 30 mM) in the bath, cells were voltage clamped while imaged confocally every 515 ms at the cell-bottom (applies if not mentioned otherwise, Fig. 1a). A 1 s depolarization (depol1s, from

80 mV to 10 mV, if not mentioned otherwise) induced a

calcium current (ICa) of 30934 pA (mean s.e.m., n 60), a

capacitance jump (DCm) of 36533 fF (n 60) and 10.61.2

uorescent A647 spots in B70160 mm2 of the foot area of the cell (n 60 cells, Fig. 1b). This stimulation protocol was used

because it consistently induces endocytosis (Fig. 1b)15,26.

Three sets of evidence suggest that A647 spots reect vesicle fusion, owing to A647 diffusion from the bath to open vesicles. First, most spots occurred during and within 1 s after depolarization (Fig. 1c, upper, 60 cells, 636 spots in total). This time course (Fig. 1c, upper) was parallel to the immediate capacitance jump induced by depolarization, which reects exocytosis (Fig. 1c, lower). The spot number per cell was also proportional to the DCm (Fig. 1d). Second, analogous to calcium-triggered exocytosis, removing extracellular calcium abolished both spot occurrence and DCm (n 10 cells, Po0.01, Supplementary

Fig. 1). Third, in cells expressing neuropeptide Y-EGFP (NPYEGFP, granule lumen cargo), 97% ( 175/181) of the NPY-EGFP

release events were accompanied with A647 spots (Fig. 1e, arrows). The remaining 3% were likely too small or fast to be resolved. 585% of A647 spots (n 350 spots, 23 cells)

overlapped with NPY-EGFP-positive granules and their release (Fig. 1e). The percentage of overlap increased linearly towards 100% as the NPY-EGFP-positive granule density increased (Fig. 1f). Thus, A647 spots that did not overlap with NPYEGFP-positive granules were due to fusion of NPY-EGFP-negative granules.

Four sets of evidence suggest that the A647 spot at the onset reects an O-prole at the fusion instant. First, under the confocal microscope, the full-width-half-maximum (WH) of NPY-EGFP-positive granule (47513 nm, n 48 granules) was similar to the

overlapping A647 spots WH at the onset (49013 nm, n 48

spots, P 0.15, t-test; Fig. 2a). STED imaging (B90 nm

resolution) conrmed that the WH of NPY-EGFP-positive vesicle (3325 nm, n 278 granules, 7 cells) was similar to the WH of

spots (36910 nm, n 101 spots, 31 cells) induced by depol1s in

different cells bathed with Alexa Fluor 488 (A488, 3060 mM, Fig. 2b). Note that A488 or EGFP, but not A647 can be used in our STED microscope. The spot WH measured with STED microscopy was close to electron microscopic measurements (B300 nm)13, supporting the idea that fused vesicles do not collapse.

Second, to visualize these structures, we conducted STED imaging at the cell-centre (42 mm above bottom), where the plasma membrane was approximately perpendicular to the coverslip (Fig. 1a). Depol1s induced A488 spots only adjacent to the cell membrane (Fig. 2c, n 45). These side images showed

O-like proles with a pore beyond our resolution (Fig. 2c).

To quantify the O-prole, we simulated an O-prole with a diameter of 300 nm and a pore o100 nm (Fig. 2d, see

Supplementary Fig. 2 for derivation). With a dye outside the cell membrane, line proles through the simulated O-prole centre showed a dip right before reaching the membrane (Fig. 2d, arrow). As expected for an O-prole, the dip for a 45 line was larger and wider than a horizontal line (Fig. 2d). These features were not observed in a simulated FC prole with an opening that is the same or larger than the vesicle diameter (Fig. 2e, Supplementary Fig. 3).

The image and line features of the simulated O-prole were also observed in STED side images of A488 spots (Fig. 2c, arrow), conrming that A488 spots were O-proles. After depolarization,

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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms4356 ARTICLE

N spot

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Figure 1 | Imaging granule fusion in chromafn cells. (a) Left: Schematic drawing showing whole-cell recording of ICa and capacitance (Cm), and imaging at the cell-bottom or centre with a uorescent dye (red) in the bath. Right: Confocal images at a real cells bottom (lower) or centre (upper) with A647 (30 mM) in the bath. The dark area at the cell-bottom represents a thin layer of A647 solution between the cell-bottom and the coverslip. (b) Left: Sampled

ICa (upper) and Cm (lower) induced by depol1s (arrow). Right: A647 confocal/cell-bottom images at 1 s before, during (0.5 s) and 10 s after depol1s (same cell as from left). Arrows indicate A647 spots. (c) The accumulated number of A647 spots (SNspot, upper) plotted versus the time at which the spots occurred in 60 cells subjected to depol1s (arrow). The corresponding mean Cm change is also plotted (lower). (d) The A647 spot number (Nspot) plotted versus Cm from the same cell (n 60, each circle represents one cell). Data were tted with a linear regression line (correlation coefcient:

0.71). (e) Concurrent imaging of NPY-EGFP (green) and A647 (red) at 0.5 s before (upper), and 0.3 s (middle) and 0.5 s (lower) during depol1s. Arrows

indicate NPY-EGFP release (green spots disappeared at 0.5 s) coincident with A647 spots. Circles indicate an A647 spot without an overlapping NPY-EGFP spot. (f) The percentage of A647 spots that are co-localized with NPY-EGFP release (spot_release) plotted versus the number of NPY-EGFP granules per mm2 at the cell-bottom (NNPY mm 2). Each circle represents one cell (n 23 cells; cells with45 A647 spots were used).

we did not observe spots that resembled the simulated collapse-like images and line features (see Methods for criteria of side imaging). However, we did observe some resting membrane curvatures (Fig. 2f) resembling the simulated collapse image and line features (Fig. 2e). Thus, the lack of collapse proles after depolarization was not due to limited spatial resolution.

Third, the O-prole appeared at the spot onset (within 36 ms, our time resolution, 45 spots, see also Figs 35). This rapid appearance, together with the overlap between the spot appearance and NPY-EGFP release (Fig. 1e,f), suggests that the O-prole is due to fusion, but not slow, endocytic membrane invagination.

Fourth, some spots may close their pore without detectable structural changes (for example, O-close fusion, described later), conrming that the O-proles pore is too small to be resolved.

Seven modes of X-prole change. To determine how O-shaped membrane proles change in size and pore opening or closing status, we performed confocal imaging (every 515 ms) at the cell-bottom with A647 and A488 in the bath being excited strongly and weakly, respectively (A647/A488 experiments, reasons explained later). We found that the structures may change in seven ways described below (636 spots, 60 cells), which reect different size changes of the O-prole (no change, enlarge, shrink partially or completely) and the pore status (opened or closed). These patterns were conrmed with STED imaging at the cell-bottom (STED/cell-bottom, every 26 ms, strong excitation) and cell-centre (STED/cell-centre, every 36 ms, strong excitation).

In 11% of the spots (73/636 spots) in confocal/A647/A488 experiments, A647 and A488 uorescence intensity (F647, F488)

peaked mostly rapidly (o100 ms) and stayed unchanged (o25%) for 30 s (the end of our image recording), during which A647 or A488 spots WH remained stable (A647: 4858 nm at onset, 4747 nm 30 s later, n 73 spots; P 0.56, t-test; Fig. 3a,

Supplementary Movie 1). Similar results were conrmed with STED/cell-bottom imaging: WH was 38035 nm at the onset, and was 40040 nm 68 s later (n 5; P 0.74; Fig. 3b). STED

data beyond B810 s after stimulation were discarded, because the whole-cell conguration was often lost due to strong STED depletion laser. STED/cell-centre imaging showed an O-prole with a dip in line proles, which remained stable for 68 s (n 6,

Fig. 3c). This pattern is termed O-stay with an open pore. The evidence supporting an open pore is explained below.

In 33% of the spots in A647/A488 experiments, F647 and F488

peaked simultaneously (o70 ms). Subsequently, F647 remained unchanged for 0.330 s, then decayed monoexponentially to baseline with a t of 2.90.1 s, whereas F488 remained unchanged (n 210 spots, Fig. 3d,e; Supplementary Movie 2). The A647 or

A488 spots WH also remained stable: A647 spots WH was 4745 nm at the F647 peak, and was 4624 nm when F647 decayed to 251% of the peak (n 210, P 0.18, t-test;

Fig. 3d,e). STED/cell-bottom imaging conrmed this result: WH was 37117 nm at the uorescence (FSTED) peak, and was

35116 nm at 2030% of FSTED peak (n 27, P 0.43, Fig. 3f).

This pattern, termed here as O-close, reects O-prole pore closure resembling KR7,8, except that closing time can be long. The following two sets of evidence support this conclusion.

First, when F647 (strong excitation) but not F488 decayed, removing A647 excitation halted F647 decay, and resuming excitation recommenced F647 decay (n 11 spots, Supplementary

Fig. 4a). After F647 decay, increasing A488 excitation increased

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Confocal (same spot) STED (different spots) A647 NPY A488

0.2 s +0.3 s

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Simulated collapse profile Collapse-like profileExample 1

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Figure 2 | Resolving the X-prole at the fusion instant. (a) Confocal images of a NPY-EGFP-positive granule before release and the A647 spot at the spot onset at the same location (upper). Normalized uorescence intensity proles (Fnorm) from dotted lines are also shown (lower, applies to b). (b) STED images (upper) of a NPY-EGFP granule and an A488 spot at the spot onset (from different cells). Line proles are also plotted (lower). (c) STED/cell centre images at 0.2 s before (left) and 0.3 s (right) during depol1s (upper). Fnorm are also shown for two lines across the spot centre, one perpendicular to the plasma membrane, the other 45 apart (applies to df). The arrow indicates the typical feature of the O-prole: a dip in the line prole that is wider and larger for the 45 line. Bright uorescence in the right side of each image represents extracellular A488, whereas dim uorescence in the left side of each image means the intracellular compartment with no A488 (applies to all plots at the STED per cell-centre setting). (d, e) Simulation showing side images and line proles (solid and dotted) before (left) and after (right) the appearance of an O-prole (d, pore size: 50 nm, vesicle size: 300 nm) or a collapsed prole (e). The arrow in d indicates the typical feature of the O-prole: a dip in the line prole which is wider and larger for the 45 line. Images are taken from Supplementary Figs 2f,g and 3c. Simulation methods are described in Supplementary Figs 2,3. (f) Two STED/cell-centre images and line proles (left, right) that resemble the presumed collapsed prole. Images were obtained in resting conditions.

F488, but followed by a monoexponential decay (n 10 spots,

Supplementary Fig. 4b). Thus, strong excitation decreased F647

(or F488). However, strong excitation alone was insufcient to cause F647 decay, because F647 may remain stable (for example, Fig. 3a) or decay at different onsets (for example, Fig. 3d,e). Thus, F647 decay must reect pore closure, which prevents exchange of bleached dye (caused by strong excitation) with uorescent dye in the bath. In contrast, a stable F647 (O-stay) reects an open pore (Fig. 3ac).

Second, as described later, endocytosis reconstructed from O-close and other close modes matched whole-cell endocytosis. Moreover, block of whole-cell endocytosis by prolonging whole-cell dialysis or inhibition of dynamin abolished close fusion. Thus, close modes reect endocytosis.

In 8% of the spots in A647/A488 experiments, F647 and F488

increased in parallel in two phases, initially within B300 ms and subsequently inB120 s (Fig. 4a). WH at the onset was 4868 nm (n 52), similar to O-stay (4858 nm, n 73,

P 0.78, t-test) or O-close (4745 nm, n 210, P 0.14). It

increased to 60012 nm at the uorescence peak (n 52,

Po0.001, Fig. 4a). STED/cell-bottom imaging showed similar patterns: WH at the onset was 39913 nm (n 8), similar to that

of O-stay (380435 nm, n 5, P 0.60) or O-close

(37117 nm, n 27, P 0.42), but increased to 50826 nm

(n 8) at the peak (Po0.01). STED/cell-centre imaging revealed

an O-prole (with a dip in line proles) and its enlargement towards the cytosol (n 6 spots, Fig. 4b, Supplementary

Movie 3). This mode is termed O-enlarge-stay. The slow time

course is not due to slow diffusion of the dye into the vesicle, but to the slow size increase, because the initial spot WH was similar to O-stay or O-close, and the dye diffuses to a granule in milliseconds27.

About 2% of the spots in A647/A488 experiments showed initial changes similar to O-enlarge-stay, but followed by F647

decay to baseline with a t (2.80.2 s, n 15) similar to that

of O-close while F488 remained unchanged (Fig. 4c). This pattern reected O-prole enlargement and closure, termed O-enlarge-close.

In 13% of the spots in A647/A488 experiments, F647 and F488

peaked rapidly (o50 ms) and subsequently decreased in parallel by 5089% with a t of 27341 ms (n 84), then stayed

unchanged for 30 s (Fig. 5a). WH at the onset was 5058 nm, similar to that of O-stay, but decreased to 3596 nm at the steady-state (n 84, Po0.001, t-test, Fig. 5a). STED/cell-bottom

imaging conrmed the spot shrinkage: WH decreased from 37926 to 23718 nm (n 22, Po0.01, t-test, Fig. 5b,

Supplementary Movie 4), and the STED uorescence intensity (FSTED) in the spots outer ring decayed faster to a lower value near baseline than the spots centre (n 22, for example, Fig. 5b).

Consistently, STED/cell-centre imaging showed that the O-prole (with a dip in line proles) shrank towards the plasma membrane without vesicle budding off, then maintained the O-prole (n 12 spots, Fig. 5c), termed O-shrink-stay.

In 14% of the spots in A647/A488 experiments, F647 and F488

showed initial patterns similar to O-shrink-stay, except that after a variable delay (0.330s) in the stay phase, F647 decayed to

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Figure 3 | X-stay and X-close fusion. (ac), O-stay. (a) F647 (red), F488 (green), WH and sampled images (average of 4) at times indicated (lines) are plotted versus time for a spot at the confocalA647/A488 setting (cell-bottom). F647 and F488 were normalized to the mean value before spot appeared (applies to all plots of F647, F488, and FSTED). Images were collected every 15 ms. (b) FSTED (STED uorescence intensity), WH, and sampled images (average of 2) at times indicated are plotted versus time for a spot at the STED/cell-bottom setting (60 mM A488 in bath). Images were collected every 26 ms. (c) FSTED, WH, sampled images (average of 8, side images of the O-prole) and their line proles (normalized to peak, Fnorm) are plotted versus time for a spot at the STED/cell-centre setting. Images were collected every 36 ms. WH was measured from the prole of a vertical line (not shown, parallel to cell membrane) across the spot centre. Solid and dotted line proles correspond to solid and dotted lines, respectively. The arrangements in a, b and c apply to all plots in Figs 36 at confocal/A647/A488, STED/cell-bottom, and STED/cell-centre setting, respectively. (df) O-close at confocal/A647/A488 (d, e) and STED/cell-bottom setting (f). Arrows indicate pore closure (apply to close fusion in Figs 36). E shows two spots (upper, lower) with different pore closing time (WH and sampled images not shown).

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Figure 4 | X-enlarge-stay and O-enlarge-close. (a,b) O-enlarge-stay at confocal/A647/A488 (a) and STED/cell-centre setting (b). (c) O-enlarge-close at confocal/A647/A488 setting.

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Figure 5 | X-shrink-stay and X-shrink-close. (ac) O-shrink-stay at confocal/A647/A488 (a), STED/cell-bottom (b) and STED/cell-centre setting (c). In a, the scale was set to see dim red images, but partly saturate the brightest red image. In b, FSTED (peak normalized, FSTED_n) in the inner circle (red) and the outer ring (between red and blue circles, blue) are also plotted, showing faster decay of blue trace and thus the spot shrinkage. (c) Left two images, average of two single images; right two images, average of eight single images. (df) O-shrink-close at confocal/A647/A488 (d), STED/cell-bottom (e) and STED/cell-centre setting (f). In e, FSTED_n in the inner circle (red) and the outer ring (between red and blue circles, blue) are also plotted to

show spot shrinkage. (f) Left two images, average of two single images; right three images, average of eight single images.

baseline with a t (2.80.1 s, n 87) similar to that of O-close

while F488 remained unchanged (Fig. 5d). The spot WH was 5097 nm at the onset, decreased to 4185 nm (n 87,

Po0.001, t-test) at the temporary stay phase, then remained unchanged as F647 decayed (4096 nm at B30% of the stay amplitude, n 87, P 0.51, t-test, Fig. 5d). STED/cell-bottom

imaging showed similar pattern: WH decreased from 35618 nm at the onset to 24412 nm (n 20, Po0.001, t-test) at the

temporary stay phase, then remained stable as FSTED decayed to baseline (25413 nm at B30% of amplitude at the transition phase, n 20, P 0.47, t-test; Fig. 5e). FSTED in the spots outer

ring decayed faster to a lower value than the spots centre in the initial shrinking phase, indicating O-prole shrinkage (n 20, for

example, Fig. 5e). Consistently, STED/cell-centre imaging showed that the O-prole (and the dip in line proles) initially shrank towards the plasma membrane, then its size stayed unchanged while FSTED decayed to baseline (n 13 spots, Fig. 5f). These

results reected the O-prole shrinkage and closure, termed O-shrink-close.

In 18% of the spots in A647/A488 experiments, F647 and F488

peaked rapidly (o50 ms), then decreased in parallel to baseline with a t (1.090.11 s, n 115, Fig. 6a,b) faster than the dye

bleaching t during O-close (2.90.1 s, n 210, Po0.001, t-test).

The decay t was mostly less than 1.5 s, and sometimes only 1550 ms (Fig. 6b). The WH decreased from 50412 nm at the onset to 36510 nm (n 115) at 231% of the peak F647 (for

example, Fig. 6a), beyond which WH was too dim to measure. STED/cell-bottom imaging conrmed this pattern: WH decreased

from 36233 nm at the onset to 22527 nm at 162% of the FSTED peak (n 19, Po0.01, t-test; Fig. 6c, Supplementary Movie

5). FSTED in the spots outer ring decayed faster than the centre (n 19, Fig. 6c), conrming O-prole shrinkage. STED/cell-

centre imaging showed shrinkage of the O-prole (and the dip in line prole) without vesicle budding off (n 8, Fig. 6d,

Supplementary Movie 6), termed O-shrink.

In summary, we described seven ways in which an O-shaped membrane prole can change, including three close modes (O-close, O-enlarge-close, O-shrink-close), three stay modes (O-stay, O-enlarge-stay, O-shrink-stay) and O-shrink (Fig. 7a). Since we limited imaging to 30 s, pore closure for stay modes beyond 30 s is possible. In addition, we observed occasional events not following these typical patterns. Atypical changes included pore closure and reopening, reected as F647 bleaching (pore closure) and sudden increase to the original level (pore reopening) while F488

remained unchanged; O-stay followed by O-shrink or O-shrink-

stay, reected as stable F647 value followed by parallel decrease of

F647 and F488; and O-enlarge-stay followed by O-shrink-stay, reected as parallel increase and then decrease of both F647 and

F488. These events reect the continuous nature and exibility of post-fusion structural changes.

No FC fusion. We did not observe FC fusion (Fig. 7b). The predicted structural change of FC would be spot enlargement while dimming at the cell-bottom, and the collapse of the O-prole (Fig. 2e,f) at side images. Neither of these structural

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a b c d

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Figure 6 | X-shrink fusion. (a) O-shrink at confocal/A647/A488 setting. (b)Distribution of the F647 decay t during O-shrink fusion at confocal/A647/ A488 setting (115 spots, 60 cells, data binned every 0.5 s). (c,d) O-shrink at STED/cell-bottom (c) and STED/cell-centre setting (d). In c, FSTED_n in the

inner circle (red) and the outer ring (blue) are also plotted to show spot shrinkage. (d) Left two images, average of two single images; right three images, average of eight single images.

changes was observed after stimulation. Thus, FC fusion was inexistent or rare. This suggestion was not due to limit in time resolution, because we imaged every 515 ms, which detected nearly all NPY-EGFP releasing granules (for example, Fig. 1e). It was also not due to the stimulus we used (depol1s), because FC fusion was absent with two other stimuli: a 2-ms depolarization train at 515 Hz for 30 s that mimicked an action potential train (48 spots, 5 cells), and high potassium application (50 mM, 53 spots, 4 cells).

Spot size changes are not caused by movement. The spot enlargement or shrinking we observed was not due to focal plane changes, because while some spots enlarged or shrank, preexisting uorescent structures 12 mm away did not change (Supplementary Fig. 5ac). Could it be due to localized (o12 mm) membrane movement that pushes the O-shaped membrane prole into the cytosol in the z axis? Four sets of evidence exclude this possibility.

First, the uorescence within B0.51 mm surrounding the spot remained unchanged at the STED cell-bottom setting (n 101

spots, for example, Supplementary Fig. 6), indicating no movement surrounding the spot. Similar results were observed using FM464 to label membrane (Supplementary Fig. 7a)28 and Atto 488 to identify fusion modes (n 75 spots, 6 cells, Supplementary

Fig. 7bd). Second, at the cell-centre setting where the movement to the cytosol could be resolved at the microscopic x/y plane (illustrated in Supplementary Fig. 8ce), we did not observe any such movements (n 45 spots; for example, Supplementary

Fig. 8fj). Third, at a z resolution of B100150 nm total internal reection uorescence microscopy (TIRF, cell-bottom setting, 30 mM Alexa 555 in bath) showed that after depol1s the spot size may shrink completely (O-shrink, 33 out of 178 spots, Supplementary Fig. 9a), shrink partially (including O-shrink-stay and O-shrink-close; 33/178 spots, Supplementary Fig. 9b), enlarge (including O-enlarge-stay and O-enlarge-close; 17/178 spots, Supplementary Fig. 9c), or remain unchanged (including

O-stay and O-close; 95/178 spots, Supplementary Fig. 9d). This result excludes movements to the cytosol of B350 nm or larger (the confocal z resolution is B350 nm) as the cause for spot size changes. Fourth, to monitor such movements at STED cell-bottom setting (A488 in bath), we switched the focal plane every 70 ms between the cell-bottom (control) and a focal plane that was 300 nm above (upper). Spots induced by depol1s were

brighter and focused at the control focal plane, but dimmer and out of focus at the upper focal plane (131 spots). When spots

dimmed and shrank completely (28 spots, Supplementary Fig. 10a) or partially (40 spots, Supplementary Fig. 10b) at the control plane, FSTED at the upper plane also decreased, but

slightly faster (Supplementary Fig. 10a,b). Thus, spot shrinking at the control plane is not due to an upward movement towards the cytosol. Similarly, all spots (21 spots) that became brighter and larger at the control plane were also brighter at the upper plane (Supplementary Fig. 10c). In conclusion, the spot size change we observed is not due to movement to the cytosol, but to actual structural changes.

Seven modes observed with other image settings. The seven modes were not only observed with the A647/A488 setting, but also with other settings, which validates our observations. When we used only one dye (A647) and excited it weakly, the spot size could remain the same (n 42 spots), increase (n 7 spots), or

decrease (to some extent or till undetectable, n 23 spots;

Supplementary Fig. 11; 7 cells), indicating that the spot size changes were not due to photo-toxicity caused by strong excitation. When we excited A488 strongly, but A647 weakly, depol1s

induced 62 spots (9 cells) showing six modes (O-stay, 9 spots; O-close, 12 spots; O-enlarge-stay, 6 spots; O-enlarge-close, 0 spots; O-shrink-stay, 9 spots; O-shrink-close, 15 spots; O-shrink, 11 spots; Supplementary Fig. 12). When we replaced A647 and A488 with Atto 655 (strong excitation/confocal) and Atto 488 (weak excitation/confocal), depol1s induced 143 spots (7 cells)

showing seven modes (O-stay, 8 spots; O-close, 45 spots; O-enlarge-stay, 7 spots; O-enlarge-close, 3 spots; O-shrink-stay, 19 spots; O-shrink-close, 31 spots; O-shrink, 30 spots; Fig. 7ci). With these settings, the percentage of each mode (obtained from smaller data sets) was roughly similar to those obtained with A647/A488 setting.

Three close modes mediate rapid and slow endocytosis. Three sets of evidence in A647/A488 experiments suggest that close fusion (O-close, O-shrink-close, O-enlarge-close) mediates whole-cell rapid (a few seconds) and slow (tens of seconds) endocytosis. First, if we assigned an upstep at every spots onset and a downstep at pore closing time (F647 decay onset) with an amplitude correction for O-shrink-close and O-enlarge-close (Fig. 8a, see Methods), the up and downstep interval in close events ranged from 0.330 s (n 312; Fig. 8b), covering both

rapid and slow endocytic time frame.

Second, summation of the up and downsteps from all spots (636 spots) yielded reconstructed net exo and endocytosis

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-exoendocytosis

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Figure 7 | Seven fusion modes conrmed by imaging with Atto 655 and Atto 488. (a, b) Schematic drawings of our model called O-exoendocytosis (a, seven modes) and the classical FC and KR model (b). Dotted arrows mean that the transition may or may not take place. (c) Atto 655 uorescence intensity (FAtto655, red), Atto 488 uorescence intensity (FAtto488, green) and sampled images (average of 510 frames) at times indicated (lines) are plotted versus time for a spot undergoing O-stay fusion. FAtto655 and FAtto488 were normalized to the mean value before spot appeared. Images were

collected every 1734 ms at the confocal cell-bottom setting with Atto 655 (strong excitation) and Atto 488 (weak excitation) in the bath. (di) Similar to c, but for spots undergoing O-close (d), O-enlarge-stay (e), O-enlarge-close (f), O-shrink-stay (g), O-shrink-close (h) and O-shrink (i).

(Nexoendo, Fig. 8c, red), which matched approximately the

corresponding whole-cell endocytosis (Fig. 8c, black, 60 cells) in both time course and amplitude. The match was also observed when we divided cells into four groups based on capacitance decay: decay to baseline within 15 s (group 1), decay by 480% in 30 s (group 2, not including group 1), decay by 3080% in 30 s (group 3) and decay by o30% in 30 s (group 4, Fig. 8d). Nexoendo

and capacitance decayed rapidly with a t of 2.8 s and 3.9 s, respectively, in group 1; but decayed slowly with a t of 8.1 and13.8 s, respectively, in group 2. In groups 34, Nexoendo

and capacitance changes were similar and did not return to

baseline. The fraction that did not decay for Nexoendo and

capacitance was similar in all groups (Fig. 8e). These results suggest that close modes mediate most whole-cell rapid and slow endocytosis.

Third, since prolonged whole-cell dialysis blocks endocytosis21,29, we used this feature to determine whether close modes cause whole-cell endocytosis. In 4 cells, depol1s induced whole-cell endocytosis and 78% (53/68 spots) close fusion within 1 min after break-in, but induced no whole-cell endocytosis, and only 3% close fusion (1/31 spots, Fig. 8f, n 4 cells) 6 min later,

suggesting that close modes cause whole-cell endocytosis.

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Exoendo reconstruction

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Figure 8 | Close modes mediate rapid and slow endocytosis of the cell. (a) Examples showing net exo and endocytosis (lower: exoendo, see also Methods) reconstructed from uorescence changes (upper: F647, F488) during O-close, O-shrink-close, O-enlarge-close, O-stay, O-shrink-stay, O-enlarge-stay and O-shrink fusion (left to right). (b) Pore closure time (from open to close, not from stimulation time to closure) distribution for three close

modes (312 spots). (c) The mean Cm (s.e.m., every 1 s, baseline subtracted, upper), Nexoendo per cell (middle upper) and ICa (lower) induced by depol1s

(to 10 mV). Cm (black) and N

exoendo (red) traces are also normalized and superimposed for comparison (middle lower). Data were from 636 spots in 60 cells. (d) Similar to c, except that data in c were divided into four groups based on Cm decay: (1) decay to baseline in 15 s (165 spots, 11 cells),(2) decay by 480% in 30 s except group 1 (101 spots, 10 cells), (3) decay by 3080% in 30 s (173 spots, 18 cells) and (4) decay by o30% in 30 s (197 spots, 21 cells). (e) The percentage of Nexoendo at 30 s after depol1s (compared with the peak Nexoendo) plotted versus the corresponding undecayed

Cm percentage at 30 s after stimulation from four groups described in d (left to right, group 14). A line (red) with a slope of 1 is also plotted. Error bars are s.e.m. The spot number and cell number are described in d. (f) The mean Cm change (upper, DCm) and the percentage of close fusion (Closesum,

lower, including O-close, O-shrink-close and O-enlarge-close) induced by depol1s within 1 min (left, 53/68 spots are close fusion) and 46 min (right, 1/31 spots is close fusion) after whole-cell break-in from the same cell (n 4 cells).

Calcium inux determines fusion modes. From group 4 to 1, ICa increased from 17525 pA (n 21 cells) to 57661 pA

(n 11 cells, Fig. 8d), close events (three close modes) increased

and became dominant, whereas the stay events (three stay modes) and O-shrink decreased (Fig. 9a). The correlation between ICa and close fusion (Fig. 9a, red), between ICa and stay fusion (Fig. 9a blue), and between ICa and O-shrink (Fig. 9a, black) are causal, because in cells showing large ICa (4450 pA during o10 ms depolarization to 10 mV), when we reduced ICa using

a 1-s depolarization to 50 mV (1028 pA, n 12 cells,

Fig. 9b), close events were nearly fully blocked (3.1%), whereas stay events and O-shrink increased (Fig. 9c, open symbols, 12 cells, 64 spots). Thus, large ICa triggered close modes, whereas low ICa promoted stay modes and O-shrink.

Dynamin mediates fusion pore closure. In control cells with an Ica 4350 pA (mean: 49954 pA, n 10 cells), depol1s induced

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whole-cell endocytosis and 73% (48/66 spots) of close fusion (Fig. 9d). With the dynamin inhibitor dynasore (80 mM, 20

30 min) in the bath in cells with an ICa 4350 pA (mean: 51541 pA, n 14 cells), depol1s induced nearly no whole-cell

endocytosis (B1030% at 30 s after depol1s, Fig. 9d) and only 8% (5/63 spots) close fusion (Fig. 9d). Similarly, including in the pipette solution the dynamin inhibitory peptide (QVPSRPNRAP, 20 mM; Tocris)30, which inhibits dynamin interaction with amphiphysin, largely blocked whole-cell endocytosis and reduced close fusion to 9% (4/45 spots) in cells with an ICa4350 pA (58573 pA, n 6). These results suggest that

dynamin mediates fusion pore closure, which in turn mediates whole-cell rapid and slow endocytosis.

X-prole retains vesicular membrane protein. To determine whether the O-shaped membrane prole, which was observed in all seven modes, holds vesicular membrane proteins from diffusion, we imaged cells expressing a vesicular membrane protein VAMP2 tagged with EGFP (VAMP2-EGFP, weak excitation). A647 (bath, strong excitation) was included for identifying fusion modes (see Methods and Supplementary Fig. 13). Depol1s

induced 66 A647 spots (14 cells) that coincided with VAMP2-EGFP spots. VAMP2-EGFP uorescence (FVAMP2) increased at

A647 spot onset owing to the pH increase upon fusion (Fig. 10)31. During O-stay, FVAMP2 and spot size remained stable (n 12

spots, Fig. 10a). During O-close, spot size remained stable (n 13

spots, Fig. 10b). Pore closure lead to F647 decay, whereas FVAMP2

decreased partially in 30 s (n 13 spots, Fig. 10b), consistent with

cavicapture that takes 100 s or longer for re-acidication15,31. During O-enlarge-stay, FVAMP2 remained stable, whereas

VAMP2-EGFP spot size increased (n 5, Fig. 10c). During

O-shrink-stay, F647 decrease (to 202%, n 9) in the shrink

phase was not accompanied by parallel FVAMP2 decrease, but a

delayed and smaller FVAMP2 decrease (to 677%, n 9, for

example, Fig. 10d). However, VAMP2-EGFP spot WH decreased in parallel with A647 spot WH (for example, Fig. 10d, n 9),

suggesting that the O-shaped membrane prevents or slows down diffusion of vesicular membrane proteins.

For O-shrink-close, A647 and VAMP2-EGFP spot size reduced in parallel (for example, Fig. 10e, n 5). F

VAMP2 did not decay during the shrink phase just like O-shrink-stay, but decayed with a delay slowly (n 5, Fig. 10e), likely due to slow re

acidication15,31. During O-shrink, FVAMP2 decreased to baseline (n 22). If shrinking was rapid, a VAMP2-EGFP diffusion cloud

was observed (Fig. 10f). If shrinking was slow, VAMP2-EGFP spot size reduced in parallel with the A647 spot size (Fig. 10g). FVAMP2 reduction signicantly lagged behind F647 reduction, reminiscent of the delay observed in O-shrink-stay and O-shrink-close (Fig. 10d,e). Thus, in all seven modes, O-shaped membrane proles may prevent or slow down diffusion of vesicular membrane proteins.

At B20 s after the spot appeared, reducing the bath pH to 5.5 by applying MES solution decreased the pH-sensitive FVAMP2

(ref. 31) to baseline for stay events (including O-stay, O-enlarge-stay and O-shrink-stay, n 9 spots,Fig. 10h, left), but not for

close events (including O-close and O-shrink-close, n 8 spots,

Fig. 10, right), conrming the pore open or close status, as determined by A647 imaging.

DiscussionWe establish a new exoendocytosis model, termed O-exo

endocytosis (Fig. 7a), where the O-prolefuison does not dilate, but

changes in seven patterns through size transformation and fusion pore closure. This model is fundamentally different from the classical FC/KR model. FC is redened as O-shrink, which merges fused vesicles with the plasma membrane by shrinking,

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Figure 9 | Strong calcium inux triggers dynamin-dependent close fusion modes and low calcium promotes stay modes and X-shrink. (a) The percentage of Closesum (including all three close modes), Staysum (including three stay modes) and O-shrink plotted versus the mean ICa in four groups described in Fig. 8d (stimulation: depol1s to 10 mV). The percentage was calculated within each group. (b) Sample ICa and Cm induced by a 1 s

depolarization to 50 mV. This cell showed an ICa of B500 pA during a 10 ms depolarization to 10 mV (not shown). (c) Re-plotting a (solid symbols),

but including data similar to those shown in b (open symbols), where 1 s depolarization to 50 mV induced the smallest ICa as compared with the

mean ICa induced by depol1s to 10 mV in groups 14. (d) The mean Cm change (upper, DCm) and the percentage of close fusion (Closesum, lower,

including O-close, O-shrink-close and O-enlarge-close) induced by depol1s in control (10 cells, 66 spots, left) and in cells bathed with 80 mM dynasore (14 cells, 63 spots, right). In both groups, cells with an ICa4350 pA were selected for analysis.

Figure 10 | X-prole retains vesicle membrane protein VAMP2. (ag) F647 (red), FVAMP2 (green), WH of A647 (red) and VAMP2-EGFP (green)spot, and sampled A647 (red) and VAMP2-EGFP (green) images (at times indicated with lines) for spots undergoing O-stay (a), O-close (b), O-enlarge-stay (c), O-shrink-stay (d), O-shrink-close (e) and O-shrink (f: rapid shrinking, diffusion cloud; g: slow shrinking, size reduction observed). Cells were expressed with VAMP2-EGFP and stimulated by depol1s with A647 in the bath. WH is not measured in f, because VAMP2-EGFP rapidly diffused into a cloud, which did not reect the O-shaped membrane prole size. VAMP2-EGFP spots appeared slightly (B50100 nm in WH) larger than corresponding

A647 spots (for example, Fig. 10a,b), because VAMP2-EGFP was located at the membrane, whereas A647 was inside the O-shaped structure. (h) The F647

and FVAMP2 changes in response to a bath pH change from 7.4 to 5.5 (upper) for spots undergoing O-stay (left) and O-close (right).

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but not dilating the O-shaped membrane prole. KR is redened as close fusion, including O-close, O-shrink-close and O-enlarge-close, which may generate different-sized vesicles. Close fusion is triggered by strong calcium inux and requires dynamin to close

the pore. Close fusion is the dominant mechanism mediating whole-cell rapid and slow endocytosis. It may also regulate vesicle size by shrinking or enlarging the O-shaped membrane prole before fusion pore closure. Consequently, the enlargement

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(O-enlarge-close) may mediate bulk endocytosis, a form of endocytosis dened as formation of large vesicles from the plasma membrane5,6. While strong calcium inux triggers close fusion, weak calcium inux facilitates stay fusion (O-stay, O-shrink-stay, O-enlarge-stay) and O-shrink. Unlike the irreversible FC model, transition between the different modes in the O-exoendocytosis model is exible (Fig. 7a). Finally, most vesicular membrane proteins are maintained in the O-shaped membrane during various modes of fusion by a mechanism that needs further investigation.

Our model may account for most observations previously interpreted with FC/KR model. For example, O-shrink not only accounts for the merging of fused vesicles with the plasma membrane previously attributed to FC, but also allows for the exibility to close the fusion pore anytime during shrinking (Fig. 7a). The pore of O-shrink fusion is much larger than 4 nm, because O-shrink released the B4 nm NPY-EGFP32 rapidly with a t of 334 ms (n 49 spots, for example, Fig. 1e). O-shrink

with a large pore may explain all live-cell data previously interpreted as FC, such as rapid content release, fusion pore conductance increase above a detection limit (corresponds to B35 nm pore), and disappearance of fusion-generated vesicular images in endocrine cells and neurons13,15,25,27,31,3337.

Could O-shrink replace FC at synapses? FC was rst suggested by Heuser and Reese2 based on freeze-fracture electron microscopic observation that there are more large openings (diameter: 60120 nm) at 50 ms than at 35 ms after stimulation at neuromuscular junctions38. It predicts that as pore dilates, large openings become dominant. However, large openings are not dominant at any time measured2. Using similar techniques and preparation, Ceccarelli et al.7 questioned whether FC exists39,40. A recent study shows a widened neck of an O-prole consistent with the FC model41. However, FC has not been observed in live cells. An increased surface area42 and release of B15 nm quantum dot43 at live retinal and hippocampal nerve terminals support FC, but can also be interpreted with O-shrink that has a large pore. Further work is needed to conrm O-shrink fusion in live synapses.

By comparing endocytosis reconstructed from 636 fusion events with concurrently measured whole-cell endocytosis, we found that fusion pore closure during close fusion mediates most slow endocytosis within 30 s after stimulation (Fig. 8c,d, group 24). We cannot exclude classical endocytosis beyond 30 s, although closure of stay fusion may also contribute to whole-cell endocytosis. Our nding challenges the traditional view that slow endocytosis is mediated by classical endocytosis.

Can close fusion contribute to slow endocytosis at synapses? Inhibition of slow endocytosis by blocking clathrin-dependent endocytosis5 and the existence of a readily retrievable pool4446 argue against this possibility. However, it remains unclear whether close fusion is clathrin-dependent and whether stay fusion generates the readily retrievable pool. Further study is needed to determine whether close fusion contributes to slow endocytosis at synapses.

Rapid endocytosis in endocrine cells and neurons is hypothesized to be caused by KR8,19,47, rapid classical endocytosis46,48 or bulk endocytosis42,49. Which mechanism mediates rapid endocytosis remains unresolved, because each endocytic modes contribution had not been reconstructed for comparison with whole-cell endocytosis. Providing such comparison for the rst time, we found that close fusion (three close modes), which includes bulk endocytosis (mediated by O-enlarge-close), underlies rapid endocytosis (Fig. 8d, group 1).

The three close modes provide a mechanism to regulate vesicle size. Hence, they may contribute to or cause vesicle size variation observed within a cell and among different cells50,51. Since vesicle

size is proportional to quantal size50,51, regulation of three close modes may in turn modulate quantal size, which denes exocytosis strength, such as increased vesicle size and synaptic strength observed after animal activity52. Previous studies suggest that low calcium concentration triggers rapid KR17,18, which predicts rapid whole-cell endocytosis when calcium inux is reduced or buffered. In contrast, reducing or buffering calcium inux abolishes endocytosis in chromafn cells and neurons20,2224,53,54. The present work may explain this discrepancy, because in studies suggesting that KR is triggered by low calcium, an indirectly detected O-prole, but not pore closure is interpreted as KR17,18. Such an interpretation is analogous to low calcium-facilitated stay modes reported here (Fig. 9). We found that strong calcium inux triggers close fusion to mediate whole-cell endocytosis, consistent with the nding that calcium inux triggers whole-cell endocytosis (Figs 8,9)2024,53,54.

Our nding that strong calcium inux triggers close fusion is apparently consistent with results showing that KR is dominant at 90 mM extracellular calcium14. However, increasing extracellular calcium (50 mM) decreases ICa during depolarization15, implying that prolonged extracellular, but not intracellular calcium increase facilitates KR. Consistent with this implication, prolonged intracellular calcium increase does not promote KR55. Taken together, we conclude that strong calcium inux induced by transient depolarization triggers close fusion.

Rapid KR (within seconds) is proposed as a simple reversal of fusion pore opening without dynamin involvement, whereas cavicapure (slow KR) may require dynamin4,56,57. The present work provides experimental data showing that not only slow, but also rapid fusion pore closure is mediated by dynamin (Fig. 9d). Our nding that close fusion is a dominant endocytic mechanism seems in contrast to the infrequent KR observed in cell-attached recordings. This difference is likely due to different denitions and recording conditions. In cell-attached recordings, KR is detected as equal sized capacitance up- and down-steps that occur within 2 s7,35,37,58,59, which would exclude O-shrink-close, O-enlarge-close and O-close that closes after 2 s. Furthermore, close modes were triggered by strong calcium inux during transient depolarization (Fig. 8), whereas KR in cell-attached recordings is often detected at rest or with high potassium depoarization13,14,35,37,59, which does not promote close fusion.

How the granule dense core copes with structural changes during O-exoendocytosis is unknown. It might be squeezed out or dissolved rst and then released during O-shrink60,61, but might stay as observed in pituitary lactotrophs61 during stay or close fusion.

In summary, the O-exoendocytosis model may explain most live-cell data previously interpreted with FC/KR model. It may apply to large vesicles in many other cell types, such as pancreatic cells, adipocytes, blood cells, glial cells and neurons that secret dopamine, peptides and hormones62. Whether it applies to small synaptic vesicles deserves consideration, because neuroendocrine chromafn cells and nerve terminals are similar in many aspects that were traditionally interpreted with classical exo and endocytosis models, such as capacitance upsteps and ickers, rapid and slow content release, calcium-triggered rapid and slow endocytosis, bulk endocytosis and proteins involved in exo and endocytosis1,4,5,10,11,20,23,24,63.

Vesicle fusion has been imaged by many techniques, including TIRF31 or polarized TIRF imaging64, two-photon imaging with extracellular dye that our technique is based on25, and interference reection microscopy42. Using confocal and STED microscopy, we achieved the highest spatial and temporal resolution currently available, B90 nm/515 ms. We detected pore closure by differential excitation of two dyes at a temporal

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resolution (B0.3 s) much faster than other imaging methods31. Without relying on protein overexpression, our method is much more efcient in capturing vesicle fusion (Fig. 1e,f). Our imaging method opens the door for studying the mechanisms that generate and regulate the O-prole, or more generally, membrane curvature.

Methods

Primary bovine chromafn cell culture. We prepared primary chromafn cell culture as described previously65. In brief, fresh adult (2127 months old) bovine adrenal glands (local abattoir), were immersed in pre-chilled 1 Locks buffer on

ice containing: NaCl, 145 mM; KCl, 5.4 mM; Na2HPO4, 2.2 mM; NaH2PO4,0.9 mM; glucose, 5.6 mM; HEPES, 10 mM; pH 7.3 adjusted with NaOH. Glands were perfused with 1 Locks buffer, then infused with Locks buffer containing

collagenase P (1.5 mg ml 1, Roche), trypsin inhibitor (0.325 mg ml 1, Sigma) and bovine serum albumin (5 mg ml 1, Sigma) and incubated at 37 C for 20 min. The digested medulla was minced in Locks buffer and ltered through a nylon mesh.

The ltrate was centrifuged (39 g, 4 min), re-suspended in Locks buffer and re-centrifuged until the supernatant was clear. Final cell pellet was re-suspended in pre-warmed DMEM low-glucose medium (Gibco) supplemented with 10% fetal bovine serum (Gibco) and plated onto poly-L-lysine (0.005% w/v, Sigma) and laminin (4 mg ml 1, Sigma)-coated glass coverslips. The cells were incubated at 37 C with 8% CO2 and used within 1 week. Before plating, some cells were transfected by electroporation (2 mg plasmid DNA containing NPY-EGFP or VAMP2-EGFP) using Basic Neuron Nucleofector Kit (Lonza, Program O-005).

Electrophysiology. At room temperature (2224 C), whole-cell voltage clamp and capacitance recordings were performed with an EPC-10 amplier together with the software lock-in amplier (PULSE, HEKA, Lambrecht, Germany)23,66. The holding potential was 80 mV. The frequency of the sinusoidal stimulus was 1,0001,500 Hz with a peak-to-peak voltage r50 mV. The bath solution contained 125 mM NaCl, 10 mM glucose, 10 mM HEPES, 5 mM CaCl2, 1 mM MgCl2, 4.5 mM KCl, 0.001 mM TTX and 20 mM TEA, pH 7.3 adjusted with NaOH. The pipette (36 MO) solution contained 130 mM Cs-glutamate, 0.5 mM Cs-EGTA, 12 mM

NaCl, 30 mM HEPES, 1 mM MgCl2, 2 mM ATP and 0.5 mM GTP, pH 7.2 adjusted with CsOH. These solutions pharmacologically isolated calcium currents.

Imaging. With an inverted confocal microscope (TCS SP5II, Leica, Germany, 100 oil objective, numerical aperture: 1.4), A647 (30 mM in bath, Invitrogen) and

A488 (3060 mM in bath, Invitrogen) were excited by a HeNe laser at 633 nm (maximum power: 20 mW) and an Argon laser at 488 nm (maximum power:25 mW), respectively. Unless mentioned otherwise, the 633 nm laser was set at 60% of the maximum power, whereas 488 nm laser was set at 1.52%. A647 uorescence was collected with a photomultiplier at 643700 nm, whereas A488, with a GaAsP hybrid detection system at 498580 nm. The quantum efciency of the hybrid detection system is two times higher than the photomultiplier, which improved the signal-to-noise ratio for A488 imaging. Both excitation and uorescence collection were done simultaneously.

The inverted STED microscope has a resolution of 80 nm (TCS STED, Leica, Germany), which (B8090 nm) was conrmed with uorescent bead measurements. A488 (60 mM) was excited with an Argon laser at 488 nm at 20% of the maximum power (maximum power: 25 mW), and depleted with a continuous wave ber laser at 592 nm using the maximum power (1.5 W). The uorescence was acquired by GaAsP hybrid detection system at 498580 nm. At 20% of the maximum power, 488 nm laser caused A488 bleaching after pore closure with a time course similar to that of A647 under the confocal setting.

Confocal or STED imaging area was B70160 mm2. Images were collected every 515 ms at 5070 nm per pixel at the confocal/A647/A488 setting, every26 ms at 40 nm per pixel at the STED/cell-bottom setting and every 36 ms at 40 nm per pixel at the STED/cell-centre setting.

For imaging at STED/cell-centre setting, we chose a focal plane that showed a clear edge between the solution and the cytosolic compartment of the cell, an indication that the cell membrane was approximately in parallel with the microscopes z axis. More specically, at such a focal plane, the distance that covered 2080% uorescence changes between the solution and the cytosolic compartment was less than 300 nm, beyond which the cell was not used. Furthermore, to clearly resolve the O-prole or the FC prole, we only analysed uorescent spots with an initial WH more than 350 nm. This selection avoided challenging our limited spatial resolution (B90 nm) when a small spot shrank until undetectable.

TIRF imaging (Olympus FV1000) was taken every 100200 ms with a 60 oil

immersion objective (NA: 1.45) and an EMCCD camera (Hamamatsu Photonics). Alexa 555 in the bath solution was excited by a HeNe laser at 543 nm.

Fluorescence measurements and presentations. Images were analysed using ImageJ. The uorescence intensity (F647, F488, FSTED) from an area covering the

uorescence spot was measured at every image frame. For images shown in gures

and movies, 24 frames were averaged at the confocal/A647/A488 setting, 23 frames were averaged at STED/cell-bottom setting and 28 frames were averaged at STED/cell-centre setting. The averaging improved signals and usually did not sacrice the time resolution, because most changes were much slower than the time needed for averaging (Figs 36). However, a fraction of shrink fusion (O-shrink, O-shrink-stay, O-shrink-close) may take only 50200 ms during the shrinking phase, in which we used 12 frames for averaging. WH was measured from intensity proles of 14 lines (for example, Fig. 2a,b) across the spot centre at 45 or 90 apart. At STED/cell-centre setting, WH was measured from line proles approximately in parallel with the cell membrane (Figs 3c, 4b, 5c,f and 6d). For line proles, we normalized the peak uorescence to 1. For F647, F488 and FSTED, the

value before the spot appeared was normalized to 1.

Identifying fusion modes with a single dye. When A647 (strong excitation) and EGFP were imaged (Fig. 10), most fusion modes except O-shrink and O-close, could be readily distinguished from F647 change alone, as shown in Figs 36. Although F647 decayed in both O-shrink and O-close fusion, the decay t for O-shrink (1.090.11 s, n 115 spots, Supplementary Fig. 13a) was much faster than

that of O-close (2.90.1 s, n 210 spots, Supplementary Fig. 13b). Since the decay,

t distribution for O-shrink overlapped little with that of O-close (Supplementary Fig. 13c), we classied spots with a decay t o1.7 s as O-shrink. This criterion excluded O-close, because the decay t for all O-close events was 41.7 s (Supplementary Fig. 13). Spots with a decay t42 s were classied as O-close, in which 8% could be due to O-shrink owing to the overlap distribution of the decay t (Supplementary Fig. 13c). Such a small error should not signicantly affect our main conclusion.

With a single dye (A488, strong excitation) in STED imaging, we identied the fusion modes based on changes in FSTED and WH similar to confocal imaging.

STED imaging was more sensitive in detecting the WH change, making it easier to distinguish between O-shrink and O-close.

Exo and endocytosis reconstruction. In confocal/A647/A488 experiments, we labelled each spot at the spot onset as an upstep with an amplitude of 1, and a downstep only for three close modes at the pore closure time, that is, the onset of A647 bleaching while A488 remained unchanged (Fig. 8a). The downstep amplitude was corrected for O-shrink-close and O-enlarge-close by raising the relative uorescence changes during the shrink or enlarge phase to a power of 2/3, which was approximately proportional to the granule surface area or capacitance. This correction was based on our observation that the uorescence change was approximately proportional to the WH change raised to a power of 3. Here we did not consider granule size differences for each fusion mode, because the mean spot WH at the fusion onset was similar for seven fusion modes.

Measuring the pore closure time. For close modes, the onset of the F647 decay when F488 remained unchanged was taken as pore closure time. The F647 decay time constant was 2.90.1 s (n 210), meaning that at 0.3 s, F647 decays by 10%,

which could be well resolved (for example, arrows in Figs 3df, 4c and 5df). Thus, our time resolution for pore closure was 0.3 s. If pore closure was less than our resolution, we assigned an arbitrary value of 0.3 s.

Data selection for analysis. The data within the rst 2 min after whole-cell break-in were used to avoid whole-cell endocytosis rundown (see also Fig. 8f)21. Cells showing prominent endocytosis overshoot were discarded, because we focused on determining whether close modes are responsible for compensatory endocytosis, and the overshoot often curtailed the capacitance increase to a negative value, making it difcult to compare whole-cell exoendo with reconstructed exo endocytosis.

Statistics. The statistical test used is t-test or paired t-test. The data were expressed as means.e.m.

References

1. Sudhof, T. C. The synaptic vesicle cycle. Annu. Rev. Neurosci. 27, 509547 (2004).

2. Heuser, J. E. & Reese, T. S. Structural changes after transmitter release at the frog neuromuscular junction. J. Cell Biol. 88, 564580 (1981).

3. Miller, T. M. & Heuser, J. E. Endocytosis of synaptic vesicle membrane at the frog neuromuscular junction. J. Cell Biol. 98, 685698 (1984).

4. Alabi, A. A. & Tsien, R. W. Perspectives on kiss-and-run: role in exocytosis, endocytosis, and neurotransmission. Annu. Rev. Physiol. 75, 393422 (2013).

5. Saheki, Y. & De Camilli, P. Synaptic vesicle endocytosis. Cold Spring Harb. Perspect. Biol. 4, a005645 (2012).

6. Wu, L. G., Hamid, E., Shin, W. & Chiang, H. C. Exocytosis and endocytosis: modes, function, and coupling mechanisms. Annu. Rev. Physiol. 76, 301331 (2014).

7. Ceccarelli, B., Hurlbut, W. P. & Mauro, A. Turnover of transmitter and synaptic vesicles at the frog neuromuscular junction. J. Cell Biol. 57, 499524 (1973).

NATURE COMMUNICATIONS | 5:3356 | DOI: 10.1038/ncomms4356 | http://www.nature.com/naturecommunications

Web End =www.nature.com/naturecommunications 13

ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms4356

8. Fesce, R., Grohovaz, F., Valtorta, F. & Meldolesi, J. Neurotransmitter release, fusion or kiss and run? Trends Cell Biol. 4, 14 (1994).

9. Hell, S. W. Far-eld optical nanoscopy. Science 316, 11531158 (2007).10. Lindau, M. & Alvarez de Toledo, G. The fusion pore. Biochim. Biophys. Acta 164, 167173 (2003).

11. Jackson, M. B. & Chapman, E. R. The fusion pores of Ca(2 )-triggered

exocytosis. Nat. Struct. Mol. Biol. 15, 684689 (2008).12. Neher, E. & Marty, A. Discrete changes of cell membrane capacitance observed under conditions of enhanced secretion in bovine adrenal chromafn cells. Proc. Natl Acad. Sci. USA 79, 67126716 (1982).

13. Albillos, A. et al. The exocytotic event in chromafn cells revealed by patch amperometry. Nature 389, 509512 (1997).

14. Ales, E. et al. High calcium concentrations shift the mode of exocytosis to the kiss-and-run mechanism. Nat. Cell Biol. 1, 4044 (1999).

15. Perrais, D., Kleppe, I. C., Taraska, J. W. & Almers, W. Recapture after exocytosis causes differential retention of protein in granules of bovine chromafn cells. J. Physiol. 560, 413428 (2004).

16. He, L. & Wu, L. G. The debate on the kiss-and-run fusion at synapses. Trends Neurosci. 30, 447455 (2007).

17. Fulop, T. & Smith, C. Physiological stimulation regulates the exocytic mode through calcium activation of protein kinase C in mouse chromafn cells. Biochem. J. 399, 111119 (2006).

18. Fulop, T., Radabaugh, S. & Smith, C. Activity-dependent differential transmitter release in mouse adrenal chromafn cells. J. Neurosci. 25, 73247332 (2005).

19. Elhamdani, A., Azizi, F. & Artalejo, C. R. Double patch clamp reveals that transient fusion (kiss-and-run) is a major mechanism of secretion in calf adrenal chromafn cells: high calcium shifts the mechanism from kiss-and-run to complete fusion. J. Neurosci. 26, 30303036 (2006).

20. Artalejo, C. R., Henley, J. R., McNiven, M. A. & Palfrey, H. C. Rapic endocytosis coupled to exocytosis in adrenal chromafn cells involves Ca2, GTP, and dynamin but not clathrin. Proc. Natl Acad. Sci. USA 92, 83288332 (1995).

21. Smith, C. & Neher, E. Multiple forms of endocytosis in bovine adrenal chromafn cells. J. Cell Biol. 139, 885894 (1997).

22. Sankaranarayanan, S. & Ryan, T. A. Calcium accelerates endocytosis of vSNAREs at hippocampal synapses. Nat. Neurosci. 4, 129136 (2001).

23. Wu, X. S. et al. Ca(2 ) and calmodulin initiate all forms of endocytosis during

depolarization at a nerve terminal. Nat. Neurosci. 12, 10031010 (2009).24. Hosoi, N., Holt, M. & Sakaba, T. Calcium dependence of exo- and endocytotic coupling at a glutamatergic synapse. Neuron 63, 216229 (2009).

25. Takahashi, N., Kishimoto, T., Nemoto, T., Kadowaki, T. & Kasai, H. Fusion pore dynamics and insulin granule exocytosis in the pancreatic islet. Science 297, 13491352 (2002).

26. Engisch, K. L. & Nowycky, M. C. Compensatory and excess retrieval: two types of endocytosis following single step depolarizations in bovine adrenal chromafn cells. J. Physiol. 506 Pt 3 591608 (1998).

27. He, L. et al. Compound vesicle fusion increases quantal size and potentiates synaptic transmission. Nature 459, 9397 (2009).

28. Hoopmann, P., Rizzoli, S. O. & Betz, W. J. Imaging synaptic vesicle recycling by staining and destaining vesicles with FM dyes. Cold Spring Harb. Protoc. 2012, 7783 (2012).

29. Renden, R. & Von Gersdorff, H. Synaptic vesicle endocytosis at a CNS nerve terminal: faster kinetics at physiological temperatures and increased endocytotic capacity during maturation. J. Neurophysiol. 98, 33493359 (2007).

30. Grabs, D. et al. The SH3 domain of amphiphysin binds the proline-rich domain of dynamin at a single site that denes a new SH3 binding consensus sequence.J. Biol. Chem. 272, 1341913425 (1997).31. Taraska, J. W., Perrais, D., Ohara-Imaizumi, M., Nagamatsu, S. & Almers, W. Secretory granules are recaptured largely intact after stimulated exocytosis in cultured endocrine cells. Proc. Natl Acad. Sci. USA 100, 20702075 (2003).

32. Palm, G. J. et al. The structural basis for spectral variations in green uorescent protein. Nat. Struct. Biol. 4, 361365 (1997).

33. Chow, R. H., von Ruden, L. & Neher, E. Delay in vesicle fusion revealed by electrochemical monitoring of single secretory events in adrenal chromafn cells. Nature 356, 6063 (1992).

34. Wang, C. T. et al. Different domains of synaptotagmin control the choice between kiss-and-run and full fusion. Nature 424, 943947 (2003).

35. Klyachko, V. A. & Jackson, M. B. Capacitance steps and fusion poresof small and large-dense-core vesicles in nerve terminals. Nature 418, 8992 (2002).

36. Nemoto, T. et al. Sequential-replenishment mechanism of exocytosis in pancreatic acini. Nat. Cell Biol. 3, 253258 (2001).

37. He, L., Wu, X. S., Mohan, R. & Wu, L. G. Two modes of fusion pore opening revealed by cell-attached recordings at a synapse. Nature 444, 102105 (2006).

38. Heuser, J. E. Review of electron microscopic evidence favouring vesicle exocytosis as the structural basis for quantal release during synaptic transmission. Q. J. Exp. Physiol. 74, 10511069 (1989).

39. Torri-Tarelli, F., Grohovaz, F., Fesce, R. & Ceccarelli, B. Temporal coincidence between synaptic vesicle fusion and quantal secretion of acetylcholine. J. Cell Biol. 101, 13861399 (1985).

40. Valtorta, F. et al. Neurotransmitter release and synaptic vesicle recycling. Neuroscience 35, 477489 (1990).

41. Watanabe, S. et al. Ultrafast endocytosis at Caenorhabditis elegans neuromuscular junctions. Elife 2, e00723 (2013).

42. Llobet, A., Beaumont, V. & Lagnado, L. Real-time measurement of exocytosis and endocytosis using interference of light. Neuron 40, 10751086 (2003).

43. Zhang, Q., Li, Y. & Tsien, R. W. The dynamic control of kiss-and-run and vesicular reuse probed with single nanoparticles. Science 323, 14481453 (2009).

44. Fernandez-Alfonso, T., Kwan, R. & Ryan, T. A. Synaptic vesicles interchange their membrane proteins with a large surface reservoir during recycling. Neuron 51, 179186 (2006).

45. Wienisch, M. & Klingauf, J. Vesicular proteins exocytosed and subsequently retrieved by compensatory endocytosis are nonidentical. Nat. Neurosci. 9, 10191027 (2006).

46. Hua, Y. et al. A readily retrievable pool of synaptic vesicles. Nat. Neurosci. 14, 833839 (2011).

47. Von Gersdorff, H. & Matthews, G. Dynamics of synaptic vesicle fusion and membrane retrieval in synaptic terminals. Nature 367, 735739 (1994).

48. Balaji, J. & Ryan, T. A. Single-vesicle imaging reveals that synaptic vesicle exocytosis and endocytosis are coupled by a single stochastic mode. Proc. Natl Acad. Sci. USA 104, 2057620581 (2007).

49. Wu, W. & Wu, L. G. Rapid bulk endocytosis and its kinetics of ssion pore closure at a central synapse. Proc. Natl Acad. Sci. USA 104, 1023410239 (2007).

50. Liu, G. Presynaptic control of quantal size: kinetic mechanisms and implications for synaptic transmission and plasticity. Curr. Opin. Neurobiol. 13, 324331 (2003).

51. Lisman, J. E., Raghavachari, S. & Tsien, R. W. The sequence of events that underlie quantal transmission at central glutamatergic synapses. Nat. Rev. Neurosci. 8, 597609 (2007).

52. Steinert, J. R. et al. Experience-dependent formation and recruitment of large vesicles from reserve pool. Neuron 50, 723733 (2006).

53. Wu, W., Xu, J., Wu, X. S. & Wu, L. G. Activity-dependent acceleration of endocytosis at a central synapse. J. Neurosci. 25, 1167611683 (2005).54. Yamashita, T., Eguchi, K., Saitoh, N., Von Gersdorff, H. & Takahashi, T. Developmental shift to a mechanism of synaptic vesicle endocytosis requiring nanodomain Ca2 . Nat. Neurosci. 13, 838844 (2010).

55. Dernick, G., Alvarez de Toledo, G. & Lindau, M. Exocytosis of single chromafn granules in cell-free inside-out membrane patches. Nat. Cell Biol. 5, 358362 (2003).

56. Graham, M. E., OCallaghan, D. W., McMahon, H. T. & Burgoyne, R. D. Dynamin-dependent and dynamin-independent processes contribute to the regulation of single vesicle release kinetics and quantal size. Proc. Natl Acad. Sci. USA 99, 71247129 (2002).

57. Holroyd, P., Lang, T., Wenzel, D., De, C. P. & Jahn, R. Imaging direct, dynamin-dependent recapture of fusing secretory granules on plasma membrane lawns from PC12 cells. Proc. Natl Acad. Sci. USA 99, 1680616811 (2002).

58. Fernandez, J. M., Neher, E. & Gomperts, B. D. Capacitance measurements reveal stepwise fusion events in degranulating mast cells. Nature 312, 453455 (1984).

59. Alvarez de Toledo, G., Fernandez-Chacon, R. & Fernandez, J. M. Release of secretory products during transient vesicle fusion. Nature 363, 554558 (1993).

60. Monck, J. R., Oberhauser, A. F., Alvarez de Toledo, G. & Fernandez, J. M. Is swelling of the secretory granule matrix the force that dilates the exocytotic fusion pore? Biophys. J. 59, 3947 (1991).

61. Angleson, J. K., Cochilla, A. J., Kilic, G., Nussinovitch, I. & Betz, W. J. Regulation of dense core release from neuroendocrine cells revealed by imaging single exocytic events. Nat. Neurosci. 2, 440446 (1999).

62. Kasai, H., Takahashi, N. & Tokumaru, H. Distinct initial SNARE congurations underlying the diversity of exocytosis. Physiol. Rev. 92, 19151964 (2012).

63. Wu, L. G., Ryan, T. A. & Lagnado, L. Modes of vesicle retrieval at ribbon synapses, calyx-type synapses, and small central synapses. J. Neurosci. 27, 1179311802 (2007).

64. Anantharam, A., Onoa, B., Edwards, R. H., Holz, R. W. & Axelrod, D. Localized topological changes of the plasma membrane upon exocytosis visualized by polarized TIRFM. J. Cell Biol. 188, 415428 (2010).

65. OConnor, D. T. et al. Primary culture of bovine chromafn cells. Nat. Protoc. 2, 12481253 (2007).

66. Lindau, M. & Neher, E. Patch-clamp techniques for time-resolved capacitance measurements in single cells. Pugers Arch 411, 137146 (1988).

Acknowledgements

We thank Dr. Fujun Luo for comments on the manuscript. We thank Drs. Carolyn Smith and Lei Xue for technical assistance in imaging. We thank Drs. Justin Taraska and

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Ronald Holz for providing NPY-EGFP and VAMP2-EGFP plasmids, respectively. This work was supported by the National Institute of Neurological Disorders and Stroke Intramural Research Program.

Author contributions

H.-C.C. and W.S. did most of the experiments, designed the experiments and participated in the writing. W.-D.Z., E.H., J.S., M.B. and P.J.W. did some experiments. A.J. and F.M. were involved in setting up optical systems and initial chromafn cell cultures. L.-G.W. designed the experiments and wrote the manuscript.

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Supplementary Information accompanies this paper at http://www.nature.com/naturecommunications

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How to cite this article: Chiang, H.-C. et al. Post-fusion structural changes and their roles in exocytosis and endocytosis of dense-core vesicles. Nat. Commun. 5:3356doi: 10.1038/ncomms4356 (2014).

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Vesicle fusion with the plasma membrane generates an Ω-shaped membrane profile. Its pore is thought to dilate until flattening (full-collapse), followed by classical endocytosis to retrieve vesicles. Alternatively, the pore may close (kiss-and-run), but the triggering mechanisms and its endocytic roles remain poorly understood. Here, using confocal and stimulated emission depletion microscopy imaging of dense-core vesicles, we find that fusion-generated Ω-profiles may enlarge or shrink while maintaining vesicular membrane proteins. Closure of fusion-generated Ω-profiles, which produces various sizes of vesicles, is the dominant mechanism mediating rapid and slow endocytosis within ~1-30 s. Strong calcium influx triggers dynamin-mediated closure. Weak calcium influx does not promote closure, but facilitates the merging of Ω-profiles with the plasma membrane via shrinking rather than full-collapse. These results establish a model, termed Ω-exo-endocytosis, in which the fusion-generated Ω-profile may shrink to merge with the plasma membrane, change in size or change in size then close in response to calcium, which is the main mechanism to retrieve dense-core vesicles.

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Title

Post-fusion structural changes and their roles in exocytosis and endocytosis of dense-core vesicles

Author

Chiang, Hsueh-cheng; Shin, Wonchul; Zhao, Wei-dong; Hamid, Edaeni; Sheng, Jiansong; Baydyuk, Maryna; Wen, Peter J; Jin, Albert; Momboisse, Fanny; Wu, Ling-gang

Pages

3356

Publication year

2014

Publication date

Feb 2014

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Nature Publishing Group

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20411723

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Scholarly Journal

Language of publication

English

DOI

https://doi.org/10.1038/ncomms4356

ProQuest document ID

1501383605

Post-fusion structural changes and their roles in exocytosis and endocytosis of dense-core vesicles

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